CN108342482B - A glioblastoma marker and its application and kit - Google Patents

Abstract

The invention discloses a glioblastoma marker METTL3 and its application. METTL3 is significantly highly expressed in glioma cells, and its expression level is significantly correlated with clinical grade, with the highest expression in glioblastoma with the highest degree of malignancy; knockdown of METTL3 expression can inhibit the growth of glioblastoma cells. growth, suggesting that METTL3 could be a target for glioblastoma therapy.

Description

Glioblastoma marker, application thereof and kit

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a tissue tumor marker related to glioblastoma and application thereof.

Background

Gliomas are the most common primary intracranial malignancies. The children and the teenagers have good onset, rapid progress and short course of disease, and can affect brainstem and cause fierce disease conditions in a short time; adult disease is long in course and slow in progression, and since there are no obvious symptoms at the beginning of the development, the tumor has increased to invade the corresponding brain area by the time the patient perceives it. And the brain tissue is damaged due to surgical resection, and glioma is often poor in prognosis due to the characteristics that the tumor is difficult to remove cleanly and infiltrate to grow. At present, glioma treatment adopts a treatment means which takes operation as a main part and takes radiotherapy and chemotherapy medicines as auxiliary parts. The early detection of tumors and the discovery of new and feasible treatment methods are urgent, and a series of researches on the glioma onset and the like are carried out, but the defects of low sensitivity, low specificity and the like of the found markers still exist. The method has important significance in finding new glioma markers and prevention targets. Gliomas have different classification criteria, which can be classified according to cell morphology, malignancy of tumor cells and location. The most common clinical practice is the classification system established by the WHO, according to which gliomas are classified in 1-4 grades, the higher the grade, the higher the degree of malignancy, and the worse the prognosis. The highest grade 4 of these is called glioblastoma.

The current treatment means only can slightly prolong the life cycle of patients, and the diagnosis needs to be confirmed by comprehensive medical history, auxiliary diagnosis, postoperative pathological diagnosis and the like, so that the development of new treatment means and diagnostic markers is an urgent need for the prevention and treatment of glioblastoma. The tumor marker kit is used for detecting the molecular pathological changes of patients, and early screening or auxiliary diagnosis is realized, so that effective tumor markers need to be found. Glioma-specific treatment by new means such as gene therapy, immunotherapy requires the finding of specific markers for glioblastoma cells.

METTL3(NCBI accession No. NM-019852.4, GeneID:56339) is an enzyme that methylates RNA and is widely expressed in human tissues, but its expression level is strictly regulated. Studies in other species have shown that METTL3 deficiency causes meiosis or dysplasia.

Disclosure of Invention

The invention screens out a tumor marker related to glioblastoma, namely METTL3 gene. The research finds that the glioma cell METTL3 is abnormally and highly expressed, and the expression is more obvious along with the increase of the glioma grade, particularly the expression is highest in the glioblastoma; high METTL3 expression was significantly associated with poor prognosis in glioblastoma patients; knockdown of METTL3 expression with shRNA significantly inhibited glioblastoma growth. The METTL3 can be used as a biomarker and a treatment target of glioma, particularly glioblastoma, and is used for auxiliary diagnosis, curative effect detection, prognosis judgment and treatment of glioblastoma, and screening of drugs for treating glioblastoma. Through retrieval, no relevant report and patent application aiming at METTL3 in glioblastoma exists at present.

The invention carries out database mining work, and analyzes the differential expression of genes and the correlation of partial clinical pathological parameters by collecting complete transcriptome data and clinical data in a TCGA (the Cancer Genome atlas) glioblastoma database and using collected clinical section samples of the glioblastoma. Screening complete glioblastoma multiforme sample information; analyzing the difference of glioblastoma METTL3 gene expression; survival curves of glioblastoma patients were analyzed. The results show that the expression difference of METTL3 in glioma patients has obvious correlation with tumors, different grades of gliomas have high expression of METTL3 compared with normal tissues, and the expression level of METTL3 also increases along with the increase of tumor grades.

The invention adopts RT-PCR, immunohistochemistry and gene knock-down methods to verify the expression characteristics of cancer markers and the influence of the expression characteristics on the growth function of glioblastoma cells. The experimental method mainly comprises the following parts:

1. the gene expression difference was verified in normal brain tissue and glioblastoma samples using RT-PCR: extracting total RNA of glioma and normal brain tissue; designing a primer, and carrying out PCR reaction; detecting the expression change of METTL3 gene in normal brain tissue and glioblastoma specimen. The results show that the case samples found high expression of METTL 3.

2. The influence of the gene on the growth function of the glioblastoma cells is verified at the cellular level: expressing shRNA knock-down gene; the effect of the gene on cell growth was verified. The results show that the glioblastoma cell growth capacity of knocking down the METTL3 gene is obviously reduced.

3. The influence of the gene on the growth function of glioma cells is verified at the overall level. The results show that the glioma growth ability of knocking down the METTL3 gene is obviously reduced.

The METTL3 gene can be used as a glioblastoma marker for glioblastoma diagnosis and prognosis judgment, and has good application prospect in developing glioblastoma target drugs.

The inventor finds that the primer for detecting METTL3 has good specificity and high accuracy for diagnosing glioblastoma. Therefore, the invention provides a kit for auxiliary diagnosis, curative effect prediction and prognosis judgment of glioma.

The kit for auxiliary diagnosis, curative effect prediction and prognosis judgment of glioma comprises a specific primer of METTL3 gene, wherein the specific primer of METTL3 gene comprises an upstream primer and a downstream primer, the sequence of the upstream primer is shown as SEQ ID No.1, and the sequence of the downstream primer is shown as SEQ ID No. 2.

The kit comprises a real-time fluorescent quantitative PCR detection kit and an immunohistochemical detection kit. Wherein the primers in the real-time fluorescent quantitative PCR detection kit are suitable for detecting SYBRGreen. In addition, the kit also comprises a standard DNA template and a PCR reaction system, wherein the PCR reaction solution in the PCR reaction system is real-time fluorescent quantitative PCR reaction solution and further comprises fluorescent dye. The real-time fluorescent quantitative PCR reaction solution comprises dNTP, Mg2+, Taq enzyme and buffer solution, wherein the fluorescent dye is SYBRGreenII, and the Taq enzyme is hot-start enzyme. The immunohistochemical detection kit comprises a standard substance control, a METTL3 antibody and a reagent related to immunohistochemical detection.

The use of the kit comprises obtaining a test sample from a tissue or cell; determining the expression level of a biomarker in the test sample; analyzing the expression level to generate a risk score, wherein the risk score can be used to provide a hierarchical diagnosis and prognosis of the subject. The test sample is fresh, frozen or paraffin-fixed embedded tissue.

The reference level is the expression level of the METTL3 gene in normal cells, i.e. normal human brain tissue cells, of the same line as the assay cells and free of cancerous changes, the assay tissue or cells being known or suspected to comprise tumor cells.

Drawings

FIG. 1 is a graph of the relationship between METTL3 expression levels and the survival curves of glioblastoma patients.

FIG. 2 shows the comparison of the expression level of METTL3 in glioma tissue and normal brain tissue in TCGA database (A), the comparison of the expression level of METTL3 in clinical glioma sample and normal brain tissue in RT-qPCR detection (B), and the expression level of METTL3 in different grades of glioma and normal brain tissue in immunohistochemistry (C).

FIG. 3 shows the effect of different shRNAs on the proliferative capacity of glioblastoma cells. The effect of changes in cell growth numbers (a) and shRNA interference on the growth of nude mouse glioma grafts (B) and differences in volume size (C) on the two tumor cell lines following METTL3 knockdown, respectively, are shown.

Detailed Description

The invention is further described with reference to the following figures and examples.

Example 1 data analysis by TCGA database the relationship between METTL3 gene expression and glioblastoma patient survival was explored.

And downloading the expression profile chip data of two groups of glioblastoma tissue samples and the survival state of clinical follow-up patients by adopting a TCGA standard method, and making a relation graph of the METTL3 expression level and the total survival rate of the glioblastoma tissue samples by utilizing statistical software.

As a result: referring to fig. 1, there is a significant correlation between METTL3 expression and glioblastoma patient survival, and overall survival for patients with high METTL3 expression is significantly lower than for patients with low METTL3 expression, suggesting that METTL3 is likely to have a cancer progression promoting effect.

Example 2: the relationship between METTL3 expression and overall survival of glioma patients was mined by a database.

The TCGA database was used to download transcriptome RNA-seq sequencing data from glioma patients and to analyze the expression level of METTL3 in different samples.

As a result: referring to fig. 2A, analysis found that METTL3 was significantly increased in glioma expression levels compared to normal brain tissue, and was considered as an early diagnostic indicator of glioma.

Example 3: RT-qPCR confirmed the expression difference of METTL3 gene in glioma samples and normal brain tissue.

The control group of 10 tissue samples was obtained from patients with traumatic brain tissue injury by measuring the expression state of METTL3 gene in normal brain tissue cells and tumor tissue of patients with gliomas, which were obtained by neurosurgical resection and confirmed by pathological diagnosis.

First, total RNA extraction of cells was performed. All manipulations and related reagents were placed on ice for manipulation. Extracting total RNA of cells by using Trizol reagent, which comprises the following steps: total RNA extraction is prepared. Cells were directly digested with Trizol, lysed, and Trizol was added. After adding Trizol, the cells were left at room temperature for 5min to be sufficiently lysed. The mixture was centrifuged at 12000rpm at 4 ℃ for 5min using a high-speed refrigerated centrifuge, and the precipitate was discarded. Adding chloroform into 200ul chloroform/ml Trizol, shaking and mixing for 15min, and standing at room temperature for 15 min. The mixture was again centrifuged at 12000g at 4 ℃ for 15min using a high-speed refrigerated centrifuge. The upper aqueous phase was then aspirated into another centrifuge tube. 0.5ml of isopropanol Trizol is added into the isopropanol and mixed evenly, and the mixture is placed for 10min at room temperature. The mixture was centrifuged again at 12000g at 4 ℃ for 10min by using a high-speed refrigerated centrifuge, and the supernatant was discarded, and RNA was deposited on the bottom of the tube. 1ml of 75% ethanol was added, the tube was gently shaken and the pellet suspended. Centrifuging at 4 deg.C and 8000g for 5min with high speed refrigerated centrifuge, discarding supernatant as much as possible, air drying residual RNA on ultra-clean bench for 3min, and adding 30ulH2O to dissolve RNA sample. The quality and concentration of RNA are determined by measuring the O.D value, and the purity of RNA can be ensured when the A260/A280 value is 1.6-1.8.

Second step, use

III cDNA was synthesized using the reverse transcription kit (Invitrogen) according to the following protocol: all manipulations and related reagents were worked on ice. Reagents were added to the PCR reaction tube at concentrations according to the instructions and finally the RNA sample was added. The reaction conditions in the PCR instrument were set as follows: the cDNA samples were obtained at 42 ℃ for 45min (reverse transcription) and at 75 ℃ for 10min (heat shock termination reaction).

Thirdly, quantitatively identifying the expression quantity of the METTL3 gene by PCR, and specifically comprising the following operation steps: all manipulations and related reagents were worked on ice. The reagents and concentrations shown in Table 1 were added to the PCR reaction tube and the cDNA sample was added last. The reaction conditions in the PCR instrument were set as follows: pre-denaturation at 94 ℃ for 10s, denaturation at 94 ℃ for 5s, annealing/extension at 60 ℃ for 34s, for 40 cycles, and using beta-Actin as a relative quantitative internal reference.

Table 1RT-PCR method for identifying expression quantity of METTL3 gene

Reactants	Reaction amount
		SYBR Greenq PCR Master mix(2X)	10μl
Forward Primer(10μM)	1μl
		Reverse Primer(10μM)	1μl
H2O	7.5μl
		cDNA	0.5μl
Total amount of	20μl

TABLE 2 primer sequences for RT-PCR identification of METTL3 gene

Numbering	Primer name	Sequence of
			SEQ ID NO：1	METTL3ForwardPrimer	CTGCTTGGTTGGTGTCAAAGG
SEQ ID NO：2	METTL3ReversePrimer	GCGAGTGCCAGGAGATAGTC

Data processing and analysis: the relative expression amount of the gene is calculated by a 2-delta-Ct method.

As a result: referring to fig. 2B, we examined the mRNA expression of METTL3 in glioma tissues and normal brain tissues, found that METTL3mRNA expression was increased in tumor tissues, and validated the database conclusions.

Example 4: the difference in the expression level of METTL3 gene in different grade glioma tissues and normal tissues was compared using immunohistochemical staining (fig. 2C).

Glioma grade I, II, III and IV patients were randomly selected, tumor tissues and paired normal tissues of another person were paraffin-embedded and sectioned, and METTL3 antibody (purchased from Abcam, Cat. ab195352) was used to immunohistochemically stain METTL3, observe expression, and count positive expression cells.

As a result: statistical analysis shows that the expression ratio of METTL3 in the tumor cells of the glioma patients is obviously higher than that of normal tissues, and the expression ratio is increased along with the increase of the grade, and the expression level of METTL3 in the grade IV glioma (glioblastoma) is the highest.

Example 5: the effect of down-regulating METTL3 expression levels on glioblast cell proliferation was compared using gene knockdown. (FIG. 3A)

Knockdown of METTL3 gene was performed using two different shRNA (shRNA sequences are shown in table 3), comparing the degree of downregulation of METTL3 expression in both cases. Glioma U87MG (ATCC number: HTB-14TM, human origin, epithelial adherent growth) and U251(Sigma, number: 09063001, human origin, epithelial adherent growth) cells are divided into three groups, wherein the group I is a control group without gene knockdown and expression inhibitor; group II is METTL3shRNA1 knockdown group; group III is the METTL3shRNA2 knockdown group. The proliferation of the tumor cells was observed 24h, 48h, 72h and 96h after the culture under the same culture conditions.

TABLE 3shRNA sequences for knocking down the METTL3 gene

As a result: in both the shRNA1 and shRNA2 groups, the number of cell lines was significantly lower than the control group compared to the control group (fig. 3A). These results demonstrate that knockdown METTL3 is able to inhibit growth of the glioblastoma cell line U87MG, U251 cells.

Example 6: METTL3 inhibiting tumor growth in gliomas

5X 10 from three groups of example 5⁶U87MG cells were inoculated subcutaneously into 6-week-old female nude mice, the first measurement was started on day 32, the tumor diameter was measured every 5 days thereafter, the volume of the tumor tissue was calculated, and the experiment was terminated on day 57 and the tumor tissue was taken out for photography.

As a result: the size of transplanted tumor volume in nude mice is shown in fig. 3B and 3C, and U87MG cells knocked down for METTL3 all grew more slowly than the tumor of control cells, and the tumor volume was significantly reduced.

In combination with the above results, METTL3 was significantly highly expressed in gliomas, and the expression level increased with increasing grade of gliomas, being highest in glioblastomas. And the conclusion is verified on the functional level. The high expression of METTL3 is reflected to have the capability of marking the process of the glioblastoma, and the METTL3 can be used as a glioblastoma marker and applied to auxiliary diagnosis, curative effect prediction and prognosis judgment of the glioblastoma.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

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Claims (2)

1. A glioma cell U87MG inhibitor, which is a shRNA whose sequence is shown in SEQ ID NO:4. 2. Use of the inhibitor according to claim 1 in inhibiting the proliferation of U87MG cells in vitro.

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